Human Adipose-derived Mesenchymal Stem Cells Alleviate Rat Osteoarthritis By Inhibiting Pyroptosis Of Chondrocytes

Osteoarthritis（OA）is a chronic joint disease characterized by progressive destruction of the articular cartilage,subchondral bone sclerosis,synovial hyperplasia,and osteophyte formation.It is a common cause of disability worldwide.Despite its high prevalence,the exact pathogenesis of OA is still not well understood and no effective disease-modifying treatment options for OA are currently available.Traditional medications reduce pain and swelling in the joint area,but they are not capable of repairing damaged cartilage or restoring cartilage homeostasis.Cartilage is the most important structural component of the knee joint,and its damage is directly involved in the occurrence and progression of OA disease.Cytokines related to the pathogenesis of OA,such as tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）,can stimulate chondrocytes to release cartilage-degrading enzymes resulting in proteoglycans and Type II collagen（Col II）digestion,thereby degrading the cartilage matrix.They also lead to the death of inflammatory chondrocytes.Pyroptosis,also known as cytoinflammatory necrosis,is a type of programmed cell necrosis mediated by gasdermin protein,which is manifested by the continuous expansion of cells leading to rupture of cell membranes and release of cellular contents.Studies have shown that pyroptosis may play an important role in the pathogenesis of OA.However,the detailed mechanism of chondrocyte pyroptosis remains to be elucidated.Cell therapy represented by mesenchymal stem cells（MSCs）has been widely used in the field of regenerative medicine in recent years,and has made some progress in the treatment of knee OA.MSCs have biological characteristics such as multi-directional differentiation,diverse plasticity,and low immunogenicity.They can regulate local inflammation,apoptosis and proliferation,and participate in tissue repair.When used in the treatment of OA,they can achieve a certain effect of delaying degenerative changes.The advantages of human adipose-derived mesenchymal stem cells（hAD-MSCs）are very prominent.They are abundant in source,easy to isolate,and have unique scalability.The literature describing the interaction between hAD-MSCs and chondrocytes is still limited,and there is no report on whether hAD-MSCs can attenuate OA development by inhibiting chondrocyte pyroptosis.In this study,we observed the therapeutic effect of intra-articular injection of hAD-MSCs on OA rats,and explored the effect and mechanism of hAD-MSCs on chondrocyte pyroptosis in vitro and in vivo.Objective:To explore the beneficial effects of hAD-MSCs on mitigating rat OA and their potential role of inhibiting pyroptosis of articular chondrocytes.To provide new insights into the mechanism of hAD-MSCs on the treatment of OA.Methods:1.Hulth method was performed to establish OA model,and the OA rats were randomly divided into model group,hAD-MSCs（0.5×10~6,1.0×10~6,2.0×10~6 cells/50μl）group and hyaluronic acid（HA,10 mg/ml）.Sham-operated animals were used as the control group.And each group wass guaranteed to be 10 animals.All animals were injected into the joint cavity once after modeling.An equal volume of normal saline was injected into the sham operation group and the model group.Six weeks after administration,the animals were sacrificed.X-ray examination and Kellgren-Lawrence（KL）score were performed to evaluate the knee joints of rats.The general structure of the knee joint surface and cartilage damage of the rats were observed under the operating microscope and evaluated by Pelletier scoring method.The pathological changes of rat knee joints were observated by HE staining and Safranin O/Fast green staining,and the modified Mankin score was performed.The levels of TNF-αand IL-1βin the synovial fluid,in the usual way,were detected by ELISA kits.The pyroptotic cells of cartilage tissues was detected by TUNEL assay.Immunohistochemistry was used to detect the changes in the expression of pyroptosis signaling proteins.Transmission electron microscopy was used to observe the morphology of cartilage pyroptosis.2.Flow cytometry and three-lineage differentiation experiments were used to identify hAD-MSCs.A two-step enzymatic digestion method was used to obtain rat articular chondrocytes,inverted phase contrast microscope was used to observe the morphological structure of chondrocytes.Toluidine blue staining and ColⅡstaining were used to identify chondrocytes.Different mass concentrations of TNF-α（5,10,20,40 ng/ml）were used to establish the pyroptosis model with TNF-α（0 ng/ml）as the control group.The influence of TNF-αon the viability of chondrocytes was detected by CCK-8 assay.And proteins of pyroptosis signal were detected by Western blot.Changes in the microstructure of chondrocytes were observed by scanning electron microscope.3.The effect of hAD-MSCs on rat chondrocyte pyroptosis was observed in a co-culture system in vitro.Western blot was used to determine the optimal ratio of hAD-MSCs and primary rat chondrocytes.The migration of chondrocytes was detected by Transwell method.The chondrocyte viability was detected by CCK-8 method.The pyroptosis of chondrocytes was detected by Annexin/PI method and TUNEL assay.The expression and m RNA levels of NLRP3,Caspase1 and GSDMD in chondrocytes was detected by immunofluorescence method and QPCR respectively.The expressions of NLRP3,Cleaved-caspase1,GSDMD,P-p65,MMP-13,ColⅡand TNFR1 in chondrocytes were detected by Western blot.ELISA kits were used to determinate the levels of IL-1βand IL-18 in culture supernatant of chondrocytes.Transmission electron microscopy was used to evaluate the organelle microstructures and metabolic activity of chondrocytes.4.Primary rat chondrocytes were transfected by small interference technology,and the optimal sequence of TNFR1 si RNA transfection was screened by Western blot.The expression of NLRP3,Cleaved-caspase1 and GSDMD after down-regulating TNFR1were detected by Western blot.ELISA method was used to detect the level of s TNFR1 in the co-culture system of hAD-MSCs and chondrocytes.Results:1.Therapeutic effect of hAD-MSCs on OA ratsEach administration group of hAD-MSCs could reduce articular osteosclerosis to varying degrees,improve joint space,decrease KL score,decrease Pelletier score,improve cartilage damage,improve the pathological state of rat knee joint,and significantly reduce the pathological score.ELISA detection of the levels of TNF-αand IL-1βin the synovial fluid of the rats suggested that the administration group could down-regulate the levels of these inflammatory factors.2.Effects of hAD-MSCs on pyroptosis of chondrocytes of OA ratsTUNEL results of tissue sections showed that the high rate of TUNEL-positive cells in OA rats was decreased after hAD-MSCs（1.0×10~6 cells/50μl）treatment.The results of immunohistochemistry showed that the expressions of NLRP3,Caspase1,GSDMD and TNFR1 in the cartilage tissues of the OA rats were increased,and the expression of TNFR2 was almost not expressed.The treatment of hAD-MSCs could significantly reduce the expressions of NLRP3,Caspase1,GSDMD and TNFR1.3.TNF-α（20 ng/ml）could induces pyroptosis of rat chondrocytes in vitroCompared with the control group,the cell viability of rat chondrocytes was gradually decreased with the increase of TNF-αconcentration and the expression of NLRP3,Caspase1,GSDMD and Phospho-NF-κB p65（P-p65）proteins increased.Furthermore,TNF-α（20 ng/ml）could up-regulate the expression of matrix metalloproteinase-13（MMP-13）and down-regulate the expression of ColⅡ.What is more,the articular chondrocytes were swollen,and the microstructure was destroyed.4.hAD-MSCs could inhibit pyroptosis of chondrocytes induced by TNF-αWestern blot results showed that the ratio of 1∶1 was the best ratio for the co-culture of hAD-MSCs and chondrocytes.Transwell assay showed that hAD-MSCs could promote migration of chondrocytes.The results of CCK-8 assay showed that co-culture with hAD-MSCs significantly improved chondrocyte viability and inhibited the damage of chondrocytes triggered by TNF-α.Annexin/PI assay and TUNEL assay suggestted that hAD-MSCs could inhibit chondrocyte pyroptosis.The results of immunofluorescence showed that hAD-MSCs could reduce the expression of NLRP3,Caspase1 and GSDMD in chondrocytes.The results of QPCR showed that hAD-MSCs could reduce the m RNA levels of NLRP3,Caspase1 and GSDMD in chondrocytes.Western blot results showed that hAD-MSCs could down-regulate the expression of NLRP3,Cleaved-caspase1,GSDMD,P-p65,MMP-13 and TNFR1 proteins,and up-regulate the expression of ColⅡin chondrocytes.The results of ELISA showed that hAD-MSCs could reduce the levels of IL-1βand IL-18 in the culture supernatant of chondrocytes.Transmission electron microscopy results demonstrated that hAD-MSCs could restore the damaged structure of chondrocytes.5.hAD-MSCs could inhibit TNF-αinduced chondrocyte pyroptosis by secreting s TNFR1Small interfering RNA technology was used to silence TNFR1 in primary rat chondrocytes.The results of Western blot showed that under the condition of extremely low expression of TNFR1,the protein levels of NLRP3,Cleaved-caspase 1 and GSDMD in chondrocytes were also at a low level after TNF-αstimulation.These data were comparable to the results of co-culturing with hAD-MSCs.It is suggested that the inhibition of chondrocyte pyroptosis by hAD-MSCs may be related to the inhibition of TNFR1 expression.The results of ELISA showed that the level of s TNFR1 in the control group and the TNF-αstimulated group was almost undetectable,while the level of s TNFR1 in the co-culture system of hAD-MSCs and chondrocytes was significantly increased.It is suggested that hAD-MSCs could secrete high level of s TNFR1 and competitively block TNF-α-induced chondrocyte pyroptosis,which may be the molecular mechanism of hAD-MSCs inhibiting chondrocyte pyroptosis.Conclusion:1.The intra-articular administration of hAD-MSCs can improve the articular cartilage damage in OA rats,reduce the joint pathological score and reduce the level of synovial fluid inflammatory factors,which has obvious therapeutic effect.2.The rate of TUNEL positive cells in cartilage tissues of OA rats increased,and the levels of pyroptosis signaling proteins such as NLRP3,Caspase1,and GSDMD were significantly increased,suggesting that chondrocyte pyroptosis is involved in the pathological process of OA.The intra-articular administration of hAD-MSCs can reduce the rate of TUNEL-positive cells,down-regulate the expressions of NLRP3,Caspase1,GSDMD and TNFR1 in cartilage tissue,and improve the morphological changes of chondrocytes in pyroptosis,suggesting that hAD-MSCs can inhibit the pyroptosis of chondrocytes.3.Co-culture of hAD-MSCs and primary rat chondrocytes can promote chondrocyte migration,enhance cell viability,inhibit the fluorescence expression and gene expression of pyroptotic molecules,inhibit the level of inflammatory factors,and protect the extracellular matrix.4.hAD-MSCs can inhibit the expression of TNF-αinduced chondrocyte pyroptosis signaling protein in vitro,and the mechanism may be related to the secretion of s TNFR1which hinders the binding of TNF-αand TNFR1.